The present invention relates to a method of preparing I-CreI meganuclease variants having a modified cleavage specificity. The invention relates also to the I-CreI meganuclease variants obtainable by said method and to their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes.
The invention also relates to nucleic acids encoding said variants, to expression cassettes comprising said nucleic acids, to vectors comprising said expression cassettes, to cells or organisms, plants or animals except humans, transformed by said vectors.
Meganucleases are sequence specific endonucleases recognizing large (>12 bp; usually 14-40 bp) DNA cleavage sites (Thierry and Dujon, 1992). In the wild, meganucleases are essentially represented by homing endonucleases, generally encoded by mobile genetic elements such as inteins and class I introns (Belfort and Roberts, 1997; Chevalier and Stoddard, 2001). Homing refers to the mobilization of these elements, which relies on DNA double-strand break (DSB) repair, initiated by the endonuclease activity of the meganuclease. Early studies on the HO (Haber, 1998; Klar et al., 1984; Kostriken et al., 1983), I-SceI (Colleaux et al., 1988; Jacquier and Dujon, 1985; Perrin et al., 1993; Plessis et al., 1992) and I-TevI (Bell-Pedersen et al., 1990; Bell-Pedersen et al., 1989; Bell-Pedersen et al., 1991; Mueller et al., 1996) proteins have illustrated the biology of the homing process. On another hand, these studies have also provided a paradigm for the study of DSB repair in living cells.
General asymmetry of homing endonuclease target sequences contrasts with the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns ORF or inteins have been shown to promote the homing of their respective genetic elements into allelic intronless or inteinless sites. By making a site-specific double-strand break in the intronless or inteinless alleles, these nucleases create recombinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or an intervening sequence at the DNA level.
Homing endonucleases fall into 4 separated families on the basis of pretty well conserved amino acids motifs [for review, see Chevalier and Stoddard (Nucleic Acids Research, 2001, 29, 3757-3774)]. One of them is the dodecapeptide family (dodecamer, DOD, D1-D2, LAGLIDADG (SEQ ID NO: 91), P1-P2). This is the largest family of proteins clustered by their most general conserved sequence motif: one or two copies (vast majority) of a twelve-residue sequence: the dodecapeptide. Homing endonucleases with one dodecapeptide (D) are around 20 kDa in molecular mass and act as homodimers. Those with two copies (DD) range from 25 kDa (230 amino acids) to 50 kDa (HO, 545 amino acids) with 70 to 150 residues between each motif and act as monomer. Cleavage is inside the recognition site, leaving 4 nt staggered cut with 3′H overhangs. Enzymes that contain a single copy of the LAGLIDADG (SEQ ID NO: 91) motif, such as I-CeuI and I-CreI act as homodimers and recognize a nearly palindromic homing site.
The sequence and the structure of the homing endonuclease I-CreI (pdb accession code 1g9y) have been determined (Rochaix J D et al., NAR, 1985, 13, 975-984; Heath P J et al., Nat. Struct. Biol., 1997, 4, 468-476; Wang et al., NAR, 1997, 25, 3767-3776; Jurica et al. Mol. Cell, 1998, 2, 469-476) and structural models using X-ray crystallography have been generated (Heath et al., 1997).
I-CreI comprises 163 amino acids (pdb accession code 1g9y); said endonuclease cuts as a dimer. The LAGLIDADG (SEQ ID NO: 91) motif corresponds to residues 13 to 21; on either side of the LAGLIDADG (SEQ ID NO: 91) α-helices, a four β-sheet (positions 21-29; 37-48; 66-70 and 73-78) provides a DNA binding interface that drives the interaction of the protein with the half-site of the target DNA sequence. The dimerization interface involves the two LAGLIDADG (SEQ ID NO: 91) helix as well as other residues.
The homing site recognized and cleaved by I-CreI is 22-24 bp in length and is a degenerate palindrome (see FIG. 2 of Jurica M S et al, 1998 and SEQ ID NO:65). More precisely, said I-CreI homing site is a semi-palindromic 22 bp sequence, with 7 of 11 bp identical in each half-site (Seligman L M et al., NAR, 2002, 30, 3870-3879).
The endonuclease-DNA interface has also been described (see FIG. 4 of Jurica M S et al, 1998) and has led to a number of predictions about specific protein-DNA contacts (Seligman L M et al., Genetics, 1997, 147, 1653-1664; Jurica M S et al., 1998; Chevalier B. et al., Biochemistry, 2004, 43, 14015-14026).
It emerges from said documents that:                the residues G19, D20, Q47, R51, K98 and D137 are part of the endonucleolytic site of I-CreI;        homing site sequence must have at least 20 bp to achieve a maximal binding affinity of 0.2 nM;        sequence-specific contacts are distributed across the entire length of the homing site;        base-pair substitutions can be tolerated at many different homing site positions, without seriously disrupting homing site binding or cleavage;        R51 and K98 are located in the enzyme active site and are candidates to act as Lewis acid or to activate a proton donor in the cleavage reaction; mutations in each of these residues have been observed to sharply reduce I-CreI endonucleolytic activity (R51G, K98Q);        five additional residues, which when mutated abolish I-CreI endonuclease activity are located in or near the enzyme active site (R70A, L39R, L91R, D75G, Q47H).        
These studies have paved the way for a general use of meganuclease for genome engineering. Homologous gene targeting is the most precise way to stably modify a chromosomal locus in living cells, but its low efficiency remains a major drawback. Since meganuclease-induced DSB stimulates homologous recombination up to 10 000-fold, meganucleases are today the best way to improve the efficiency of gene targeting in mammalian cells (Choulika et al., 1995; Cohen-Tannoudji et al., 1998; Donoho et al., 1998; Elliott et al., 1998; Rouet et al., 1994), and to bring it to workable efficiencies in organisms such as plants (Puchta et al., 1993; Puchta et al., 1996) and insects (Rong and Golic, 2000; Rong and Golic, 2001; Rong et al., 2002).
Meganucleases have been used to induce various kinds of homologous recombination events, such as direct repeat recombination in mammalian cells (Liang et al., 1998), plants (Siebert and Puchta, 2002), insects (Rong et al., 2002), and bacteria (Posfai et al., 1999), or interchromosomal recombination (Moynahan and Jasin, 1997; Puchta, 1999; Richardson et al., 1998).
However, this technology is still limited by the low number of potential natural target sites for meganucleases: although several hundreds of natural homing endonucleases have been identified (Belfort and Roberts, 1997; Chevalier and Stoddard, 2001), the probability to have a natural meganuclease cleaving a gene of interest is extremely low. The making of artificial meganucleases with dedicated specificities would bypass this limitation.
Artificial endonucleases with novel specificity have been made, based on the fusion of endonucleases domains to zinc-finger DNA binding domains (Bibikova et al., 2003; Bibikova et al., 2001; Bibikova et al., 2002; Porteus and Baltimore, 2003).
Homing endonucleases have also been used as scaffolds to make novel endonucleases, either by fusion of different protein domains (Chevalier et al., 2002; Epinat et al., 2003), or by mutation of single specific amino acid residues (Seligman et al., 1997, 2002; Sussman et al., 2004; International PCT Application WO 2004/067736).
The International PCT Application WO 2004/067736 describes a general method for producing a custom-made meganuclease derived from an initial meganuclease, said meganuclease variant being able to cleave a DNA target sequence which is different from the recognition and cleavage site of the initial meganuclease. This general method comprises the steps of preparing a library of meganuclease variants having mutations at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target, and selecting the variants able to cleave the DNA target sequence. When the initial meganuclease is the I-CreI N75 protein a library, wherein residues 44, 68 and 70 have been mutated was built and screened against a series of six targets close to the I-CreI natural target site; the screened mutants have altered binding profiles compared to the I-CreI N75 scaffold protein; however, they cleave the I-CreI natural target site.
Seligman et al., 2002, describe mutations altering the cleavage specificity of I-CreI. More specifically, they have studied the role of the nine amino acids of I-CreI predicted to directly contact the DNA target (Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70). Among these nine amino acids, seven are thought to interact with nucleotides at symmetrical positions (S32, Y33, N30, Q38, R68, Q44 and R70). Mutants having each of said nine amino acids and a tenth (T140) predicted to participate in a water-mediated interaction, converted to alanine, were constructed and tested in a E. coli based assay.
The resulting I-CreI mutants fell into four distinct phenotypic classes in relation to the wild-type homing site:                S32A and T140A contacts appear least important for homing site recognition,        N30A, Q38A and Q44A displayed intermediate levels of activity in each assay,        Q26A, R68A and Y33A are inactive,        K28A and R70A arc inactive and non-toxic.        It emerges from the results that I-CreI mutants at positions 30, 38, 44, 26, 68, 33, 28 and 70 have a modified behaviour in relation to the wild-type I-CreI homing site.        
As regards the mutations altering the seven symmetrical positions in the I-CreI homing site, it emerges from the obtained results that five of the seven symmetrical positions in each half-site appear to be essential for efficient site recognition in vivo by wild-type I-CreI: 2/21, 3/20, 7/16, 8/15 and 9/14 (corresponding to positions −10/+10, −9/+9, −5/+5, −4/+4 and −3/+3 in SEQ ID NO:65). All mutants altered at these positions were resistant to cleavage by wild-type I-CreI in vivo; however, in vitro assay using E. coli appears to be more sensitive than the in vivo test and allows the detection of homing sites of wild-type I-CreI more effectively than the in vivo test; thus in vitro test shows that the DNA target of wild-type I-CreI may be the followings: gtc (recognized homing site in all the cited documents), gcc or gtt triplet at the positions −5 to −3, in reference to SEQ ID NO:65.
Seligman et al. have also studied the interaction between I-CreI position 33 and homing site bases 2 and 21 (±10) or between I-CreI position 32 and homing site bases 1 and 22 (±11); Y33C, Y33H, Y33R, Y33L, Y33S and Y33T mutants were found to cleave a homing site modified in positions ±10 that is not cleaved by I-CreI (Table 3). On the other hand, S32K and S32R were found to cleave a homing site modified in positions ±11 that is cleaved by I-CreI (Table 3).
Sussman et al., 2004, report studies in which the homodimeric LAGLIDADG (SEQ ID NO: 91) homing endonuclease I-CreI is altered at positions 26, and eventually 66, or at position 33, contacting the homing site bases in positions ±6 and ±10, respectively. The resulting enzymes constructs (Q26A, Q26C, Y66R, Q26C/Y66R, Y33C, Y33H) drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro.
The overall result of the selection and characterization of enzyme point mutants against individual target site variants is both a shift and a broadening in binding specificity and in kinetics of substrate cleavage.
Each mutant displays a higher dissociation constant (lower affinity) against the original wild-type target site than does the wild-type enzyme, and each mutant displays a lower dissociation constant (higher affinity) against its novel target than does the wild-type enzyme.
The enzyme mutants display similar kinetics of substrate cleavage, with shifts and broadening in substrate preferences similar to those described for binding affinities.
To reach a larger number of DNA target sequences, it would be extremely valuable to generate new I-CreI variants with novel specificity, ie able to cleave DNA targets which are not cleaved by I-CreI or the few variants which have been isolated so far.
Such variants would be of a particular interest for genetic and genome engineering.